PPE Export by the Essential ESX-5 Secretion System in M. tuberculosis Virulence

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PROJECT SUMMARY/ABSTRACT Mycobacterium tuberculosis (Mtb) requires the ESX-5 Type VII protein secretion system for in vitro growth. Because ESX-5 is essential, the role that ESX-5 plays in Mtb virulence and the proteins exported via ESX-5 that support Mtb replication remain poorly characterized. In the related pathogen Mycobacterium marinum, ESX-5 exports many proteins of the PE and PPE protein families that are unique to the mycobacteria. Several Mtb PE and PPE proteins localize to the cell wall and were recently implicated in acquisition of diverse nutrients. The prevailing model of PE and PPE secretion suggests they are exported as heterodimers that pair uniquely and that export is promoted by an EspG chaperone that targets the PE-PPE pair to the cognate ESX secretion system for export. This proposal will test the central hypothesis that Mtb requires ESX-5 activity and a subset of ESX-5 substrates that are exported independently of the EspG5 chaperone for virulence. This hypothesis is based on preliminary data using Mtb strains in which EccD5, an ESX-5 secretion machinery subunit, or the EspG5 chaperone can be conditionally depleted. Depletion of EccD5 but not EspG5 limited Mtb growth in macrophages and on certain carbon sources. Specific Aim 1 will determine the role of ESX-5 and the EspG5 chaperone in Mtb virulence using conditional depletion of EccD5 or EspG5 and both macrophage and mouse aerosol infection models. Specific Aim 2 will use the ESX-5 conditional depletion strains to identify PPE proteins that require ESX-5 activity but not the EspG5 chaperone for export and connect ESX-5 activity to use of specific nutrients. Specific Aim 3 will examine functional redundancy between PE proteins in facilitating export of certain PPE proteins and promoting Mtb virulence using the mouse aerosol infection model. The proposed research is conceptually innovative because it challenges existing models of ESX-5 substrate export by suggesting promiscuous pairing between PE and PPE proteins and export of certain PPE proteins independent of the EspG5 chaperone. The proposed research is significant because it is expected to show that Mtb requires ESX-5 activity for pathogenesis. This knowledge would establish ESX-5 as a strategic target for development of new anti-tubercular drugs and support inclusion of ESX-5-secreted antigens in new tuberculosis vaccines.